Lectin histochemistry of nephroblastoma (Wilms' tumour)

Summary Paraffin sections of seven cases of nephroblastoma and one case of clear cell sarcoma were stained with a battery of eleven lectins conjugated to horseradish peroxidase. Lectin staining revealed similarities between blastema and stroma with respect to their content of glycoconjugates whereas blastema and epithelial cells exhibited major differences. In general, blastema and stroma contained glycoconjugates with terminal or penultimate -galactose, glycoconjugates having either biantennary or triantennary N-linked sugar chains or both, sialoglycoconjugates, and occasionally glycogen. Epithelial cells also showed these complex carbohydrates but stained additionally for terminal disaccharide galactose-(13)-N-acetylgalactosamine, terminal -galactose and terminal -N-acetylgalactosamine. Furthermore, staining with three fucose-binding lectins revealed that the linkage between terminal -fucose residues to the constituent oligosaccharide chains varied between epithelial cells, blastema and stroma. In general, the distribution and content of glycoconjugates in tumour cells comprising clear cell sarcoma resembled that in blastema and stroma of nephroblastoma. Other findings included differences in content of glycosubstance between cuboidal and columnar cells within the same tumour. Also observed were variations between a primary tumour and its metastasis with respect to the occurrence of certain complex carbohydrates.     

Lectinhistochemical demonstration of galactose- and N-acetyl-d-galactosamine residues in cells of the rat anterior pituitary

Summary Lectins are a useful tool for identification of differently glycosylated hypophyseal hormones, prohormones and glycoconjugates without hormone function. -d-galactose and -N-acetyl-d-galactosamine (GalNAc) containing glycoconjugates were identified by light microscopy with biotinylated lectins in immunocytochemically localized cells of the anterior pituitary of the rat. Galactose, histochemically detectable by the peanut lectin (PNA), was found at penultimate position of the carbohydrate chain after removal of sialic acid. Galactose containing cells correspond to gonadotrophs and thyrotrophs located mainly in medioanterior regions of the pituitary. The lectins from the soybean (SBA) and horse gram (DBA) both specific for GalNAc residues, are bound to round and also polygonal cells corresponding again to gonadotrophs and thyrotrophs.     

Is haemoglobin Gα Philadelphia linked to α-thalassaemia?

Summary The question, Is Hb G Philadelphia linked to -thalassaemia? was first posed because the abnormal haemoglobin is found in heterozygotes at a concentration greater than 25%, the proportion predicted from a 4 -chain gene model. Globin chain biosynthesis was studied in a West Indian family in which one parent had ⁺ thalassaemia and the other was heterozygous for the G Philadelphia chain gene. The former had a globin chain production ratio / well above 1, while the latter had a ratio significantly less than 1. One child of the marriage had inherited the ⁺ thallassaemia from one parent and the G Philadelphia chain gene from the other and showed the typical picture of /-thalassaemia (/ ratio slightly above normal). It is explained in the discussion that the evidence favours a close linkage of 2 -chain genes.     

Resolution of sensory and mucoid glycoconjugates with terminal α-galactose residues in the mucomicrovillar complex of the vomeronasal sensory epithelium by dual confocal laser scanning microscopy

The organization of the mucomicrovillar complex of the vomeronasal sensory epithelium of adult rats was examined using confocal laser scanning microscopy. In specimens labeled with the FITC-conjugated isolectin B₄ of Bandeiraea simplicifolia, which recognizes terminal -galactose sugar residues of glycoconjugates, we demonstrated that the mucomicrovillar complex was composed of islet-like structures with a high-density -galactose core. The mucomicrovillar complex was further resolved into sensory and mucoid components in double-labeling and dual scanning experiments. The sensory component, which consists of the dendritic terminals of olfactory marker protein-immunoreactive vomeronasal receptor neurons, contained cytosolic glycoconjugates with terminal -galactose sugar residues. The extracellular mucoid component consisted of glycoconjugates containing terminal -galactose derived from the glands associated with the vomeronasal organ. These results demonstrated the complex microchemical organization of the sensory and mucoid components of the mucomicrovillar complex.     

Sialoglycoderivatives of bovine submandibular gland identified in situ by histochemical techniques combined with lectins

Summary We employed sialidase procedures followed by lectin stainings combined with oxidizing and deacetylating agents to visualize the distribution and sequentiate sialoglycoconjugates in the bovine submandibular gland. In particular we evidenced in acinar and ductal cells the dishomogeneous presence of sialic acids acetylated in the polyhydroxy side chain (C₇, C₈, C₉), whereas O-acetyl substituents at position C₁ and/or C₄ were not found. Sialoglycoderivatives were also differentiated by the occurrence of penultimate sugars; indeed the dimers sialic acid-(23,6)--galactose and sialic acid-(26)--N-acetylgalactosamine were identified. Using such technique we supported further the possibility to develop methods for the identification of the positions of Oacetyl groups and the reconstruction of terminal disaccharides within surface and cytoplasm glycoconjugates.     

Uptake and Retention of Selenite and Selenomethionine in cultured K-562 cells

The selenium uptake and retention have been studied in K-562 cells exposed to selenite or selenomethionine. In the uptake experiments the cells were exposed to two doses of selenite (5 or 50 M) or selenomethionine (10 or 50 M). In the retention study the cells were treated for 2 h with the above mentioned doses of the selenocompounds before being observed at different times. The selenium uptake in cells exposed to selenite 5 M began to saturate at 8 h, but increased again between 48 and 96 h. In cells exposed to selenite 50 M the selenium uptake never reached a maximum, however, at 48 and 96 h the cell viability decreased strongly. The two doses of selenite showed different retention patterns, with a relatively small cellular decrease of selenium after treatment with selenite 5 M compared to treatment with 50 M of selenite. The selenium uptake in cells exposed to selenomethionine 10 M or selenomethionine 50 M began to saturate at 24 h and 48 h, respectively. The retention patterns were similar for both selenomethionine doses with a continuous decrease of the selenium concentration during the whole observation period. The results indicated a more controlled uptake and retention pattern of selenomethionine compared to selenite.     

Proteolytic digestion of α-lactalbumin: Physiological implications

The kinetics of the partial digestion of bovine -lactalbumin (-LA) by trypsin, -chymotrypsin, and pepsin was monitored by lactose synthase activity, HPLC, and difference spectrophotometry. The relative stabilities of the various metal-bound states of -LA to trypsin and chymotrypsin at 37 and 5°C decrease in the following order: Ca(II)--LA>Zn(II), Ca(II)--LA>apo--LA. The HPLC digestion patterns of Ca(II)--LA and Zn(II), Ca(II)--LA at 5 and 37°C were similar, while the corresponding digestion patterns for apo--LA were quite different, reflecting the existence of the thermally induced denaturation states of apo--LA within this temperature region. Occupation of the first Zn(II)-binding site in Ca(II)-loaded -LA slightly alters the HPLC digestion patterns at both temperatures and accelerates the digestion at 37°C due to Zn(II)-induced shift of the thermal transition of -LA, exposing some portion of thermally denatured protein. The results suggest that the binding of Zn(II) to the first Zn(II)- (or Cu(II))-specific site does not cause any drastic changes in the overall structure of -LA. The acidic form of -LA (atpH 2.2 and 37°C) was digested by pepsin at rates similar to that for the apo- or Cu(II), Ca(II)-loaded forms by trypsin or -chymotrypsin at neutralpH. Complexation of -LA with bis-ANS affords protection against pepsin cleavage. It is suggested that the protective effects of similar small lipophilic compounds to -LA may have physiological significance (e.g., for nutritional transport).On leave from the Institute of Biological Physics, USSR Academy of Sciences, Pushchino, Moscow Region, 142292, USSR.     

Lectin cytochemistry of cell types in human and canine pituitary

Summary Labeled lectins specific for different sugars were employed to identify different cell types in pituitaries from six human autopsies and seven dogs. To determine the lectins bound by each cell type, fixed-paraffin embedded sections serial to those stained with lectins were immunostained for specific hormones and the serial pairs were examined in a comparison microscope. In human pituitaries corticotrophs stained selectively with lectins having affinity for -l-fucose and the core region of complex type N-glycosyl-proteins. Some corticotrophs also stained for the presence of terminal -galactose. Thyrotrophs stained selectively with a periodate oxidation-borohydride reduction-concanavalin A sequence. Some mammotrophs evidenced content of glycoconjugate with terminal -galactose. Dendritic cells stained selectively for abundant glycogen with the periodate-reduction-concanavalin A sequence and a lectin from Griffonia simplicifolia. Adenohypophyseal cells of dog pituitary differed in showing absence of terminal -galactose in corticotrophs, presence of terminal -galactose in thyrotrophs, presence of glycoconjugate with N-glycosidically bound oligosaccharide in thyrotrophs and gonadotrophs and presence of terminal -galactose with a different lectin affinity in mammotrophs. The main contributions of lectin histochemistry applied to the pituitary include providing an additional histologic method for identification of some cell types, and localizing glycosylated prohormone or other biochemically unrecognized non-hormone glycoconjugates whose function in pituitary cells remains to be explained.This research was supported by NIH Grants AM-10956 and HL-29775 and United Health and Medical Research Foundation of South Carolina, Inc. Grant #79     

Co-replication of satellite DNA of Chironomus melanotus with mainband DNA during polytenization

The DNAs of five Chironomus species, C. plumosus, C. nuditarsis, C. thummi thummi, C. melanotus, and Camptochironomus pallidivittatus, were investigated in analytical neutral isopycnic CsCl density gradients. DNA was isolated both from larval brains (diploid-DNA) and salivary glands (polytene-DNA). The buoyant densities of mainband DNAs were 1.692 g/cm³, with the exception of C. melanotus whose mainband had a density of 1.693 g/cm³. The densities correspond to a calculated GC content of 33% and 34% respectively. Only in C. melanotus was the DNA clearly resolved into mainband DNA and two distinct satellite shoulders: Satellite I, with a buoyant density of 1.684 g/cm³ (25% GC, calculated) and satellite II of 1.679 g/cm³ (19% GC, calculated). The two satellites comprise 15% of the total DNA in the diploid-DNA and they also occur in the polytene-DNA, where they make up 11%. The results are discussed in the general context of under- and overreplication in polyploid and polytene cells.     

Ultrastructure of a cave-wall cyanophyte-Gloeocapsa NS4

Gloeocapsa strain NS4, a cyanophyte (cyanobacterium) which grows in low light levels inside cave entrances, was studied in the electron microscope by thin sectioning and freeze-etching. The cells are surrounded by a microfibrillar sheath divided by dense lamellae, which are probably an acidic mucopolysaccharide. Inside this is a typical Gramnegative cell wall. Double-replica freeze-fracture showed that the outer envelope of the wall fractures to give two faces each consisting of densely-packed particles; the particles of the outer leaflet seem to consist of subunits arranged in a hollow cylinder. A structural model of the outer envelope is proposed. The plasma membrane fractures to give a PF face with 3000 9 nm particles m^-1 and an EF face with 150–700 11–12 nm particles m^-1. The thylakoids are arranged in a pattern not previously found in a unicellular cyanophyte, parallel arrays which intersect, and may fuse with, the plasma membrane. The thylakoid membranes have 2,850 particles m^-1, mean size 10.9 nm, on the PF face and 560 particles m^-1, mean size 12.3 nm, on the EF face. Phycobilisomes are difficult to see, but may be unusually large. These ultrastructural features may be adaptations to a very low light habitat.     

On the signal transducing mechanisms involved in the synergistic interaction between interleukin-1 and bradykinin on prostaglandin biosynthesis in human gingival fibroblasts

Recombinant human interleukin-1 (IL-1) and bradykinin (BK) synergistically stimulate prostaglandin E₂ (PGE₂) formation in human gingival fibroblasts cultured for 24 h. Neither BK or IL-1 per se, nor their combinations, caused any acute stimulation of cellular cyclic AMP accumulation. BK, but not IL-1, caused a rapid, transient rise of intracellular Ca²⁺ concentration ([Ca²⁺]_i), as assessed by recordings of fura-2 fluorescence in monolayers of prelabelled gingival fibroblasts. IL-1 did not change the effect of BK on [Ca²⁺]_i. Ionomycin and A 23187, two calcium ionophores, synergistically potentiated the stimulatory effect of IL-1 on PGE₂ formation. Three different phorbol esters known to activate protein kinase C also synergistically potentiated the action of IL-1 on PGE₂ formation. Exogenously added arachidonic acid significantly enhanced the basal formation of PGE₂. In IL-1 treated cells, the enhancement of PGE₂ formation seen after addition of arachidonic acid, was synergistically upregulated by IL-1. These data show that i) the synergistic interaction between IL-1 and BK on PGE₂ formation is not due to an effect linked to an upregulation of cyclic AMP or [Ca²⁺]_i; ii) the signal transducing mechanism by which BK interacts with IL-1, however, may be linked to a BK induced stimulation of [Ca²⁺]_i and/or protein kinase C; iii) the mechanism involved in the action of IL-1 may, at least partly, be due to enhancement of the biosynthesis of prostanoids mediated by an upregulation of cyclooxygenase activity.     

Characterization of a second carotenoid β-hydroxylase gene from Arabidopsis and its relationship to the LUT1 locus

Xanthophylls are oxygenated carotenoids that perform critical roles in plants. -carotene hydroxylases (-hydroxylases) add hydroxyl groups to the -rings of carotenes and have been cloned from several bacteria and plants, including Arabidopsis. The lut1 mutation of Arabidopsis disrupts -ring hydroxylation and has been suggested to identify a related carotene hydroxylase that functions specifically on -ring structures. We have used library screening and genomics-based approaches to isolate a second -hydroxylase genomic clone and its corresponding cDNA from Arabidopsis. The encoded protein is 70% identical to the previously reported Arabidopsis -hydroxylase 1. Phylogenetic analysis indicates a common origin for the two proteins, however, their different chromosomal locations, intron positions and intron sizes suggest their duplication is not recent. Although both hydroxylases are expressed in all Arabidopsis tissues analyzed, -hydroxylase 1 mRNA is always present at higher levels. Both cDNAs encode proteins that efficiently hydroxylate the C-3 position of -ring containing carotenes and are only weakly active towards -ring containing carotenes. Neither -hydroxylase cDNA maps to the LUT1 locus, and the genomic region encompassing the LUT1 locus does not contain a third related hydroxylase. These data indicate that the LUT1 locus encodes a protein necessary for -ring hydroxylation but unrelated to -hydroxylases at the level of amino acid sequence.     

Oligodendrocytes in the normal and chronically de-afferented lateral geniculate body of the monkey

Summary Electron microscopical examination of the norma and de-afferented laterall geniculate body of the monkey following paraformaldehyde-glutaraldehyde vascular perfusion revealed distinctive morphological features of different types of oligodendrocyte. These cells were normally situated as perineuronal satellites or in relation to axons and capillaries. A wide range of nuclear and cytoplasmic densities were displayed by both satellite and interfascicular oligodendrocytes. The following distinctive features for the identification of ligodendrocytes were utilised: the presence of large quantities of free ribosomes and ribosomal rosettes, microtubular profiles, dense marginal aggregation of nuclear chromatin together with light patches and numerous nuclear pores; but the absence of broad cytoplasmic processes, glycogen and gliofibrils. Circumferential perinuclear organization of the cytoplasmic organelles was typical of oligodendrocytes. Particular attention was paid to perineuronal satellite cells in view of the known transneuronal atrophy in the de-afferented geniculate body. Some cells having a nuclear pattern of oligodendrocytes but showing hyalinisation of perikaryon were seen in de-afferented laminae. A notable feature was the presence of variegated osmiophilic bodies in the perikaryon of oligodendrocytes also situated in the de-afferented laminae. A cell type combining the features of oligodendrocytes and astrocytes was classified as intermediate neuroglia.Fellow of the Alexander von Humboldt Foundation, on Sabbatical leave from J. Nehru Medical College Aligarh, India.Recipient of the Deutsche Forschungsgemeinschaft Grant No. G./28/15.     

Observations on the fine structure of the cercaria of Notocotylus attenuatus and formation of the cyst wall of the metacercaria

Summary The dorsal tegument of the mature cercaria of Notocotylus attenuatus is a syncytial, cytoplasmic layer, containing two types of secretory granule which are identifiable ultrastructurally. The type 1 secretory bodies are electron lucid, whereas most type 2 granules have a banded appearance. The ventral tegument contains granules which are secreted from the type 3 cells; the type 3 granules are membrane bound, electron dense, and consist of both an amorphous and a finely striated zone. The type 4 cells mainly contain cigar-shaped granules consisting of an amorphous core surrounded by concentric striations. The granules exhibit structural variability in shape and content. The type 4 cells undergo a cellular migration to the tegument during encystment. The structure of the posterior-lateral glands and mode of secretion of the granules are described. Possible functions of microtubules are discussed for each cell type. Details of some secretory processes involved in the formation of the hemispherical cyst wall are described. The layers of the cyst wall may be related to the granular contents of the various parenchymal cells of the cercaria. The tegument of the metacercaria originates primarily from the cytoplasm of the type 1, type 2, type 3 and type 4 cells.     

A manganese requirement for patulin biosynthesis by cultures of Penicillium urticae

Antibiotic production by submerged cultures of Penicillium urticae required manganese supplementation of the media. Thus, manganese supplementation (152 M) allowed accumulation of patulin to high concentrations (2 mol/mL), whereas manganese deficiency (1.53 M) resulted in the accumulation to similar levels of the first committed pathway intermediate, methyl-salicylic acid, without significant patulin accumulation. Preliminary studies suggest that a similar manganese effect may occur in other fungal species.     

Normal meiosis in two 47,XYY men

Summary Details of testicular histology and meiosis are given for two 47,XYY men, one an oligospermic childless individual, the other a fertile man with near-normal spermatogenic activity in his testes. Examination of the chromosomes at meiosis, with Q and C staining, gave no evidence for the occurrence of the second Y chromosome in the germ line of either individual.     

Adventitious shoot regeneration from in vitro leaves of several pear cultivars (Pyrus communis L.)

An efficient adventitious shoot regeneration system was developed for pear (Pyrus communis L.), using leaves from in vitro proliferating shoots. Under optimal conditions, bud regeneration frequencies of Comice, Passe-Crassane, Williams and Conference ranged from 60% to 97%, with the mean number of shoots per regenerating leaf ranging from 3.2 to 6.6. Despite the great variability in responses of the different cultivars, in general an initial dark exposure of at least 20 days was required. Ammonium and total nitrogen proved to play an essential role: intermediate NH₄ ⁺ concentrations were suitable for regeneration. The balance between NH₄ ⁺ and NO₃ ^- also influenced regeneration; optimal regeneration occured on media with a 1:3 NH₄ ⁺/NO₃ ^- ratio. TDZ at 1 M was less efficient than higher concentrations, whatever the NAA level. Finally, length and growth regulator composition of the two phases (induction and expression) influenced the regeneration rate of Conference.Abbreviations BA 6-benzyladenine - EDFS ethylenediamine-tetraacetic acid ferric-sodium salt - IBA 4-indole-3yl-butyric acid - NAA -naphthaleneacetic acid - TDZ thidiazuron (N-phenyl-N-1,2,3-thidiazol-5-ylurea)     

cDNA cloning of β-tubulin gene and organization of tubulin genes in Vigna radiata (mung bean) genome

We isolated an almost full-length cDNA clone containing -tubulin gene from a partial cDNA library of mung bean using chicken cDNA as probe. Cross-hybridization with chicken -tubulin cDNA and positive hybridization-selection and translation of mung bean mRNA established that this clone contains -tubulin sequences. We studied the organization of tubulin genes in mung bean. In this plant tubulin genes are organized in tandem repeats of alternating - and -tubulin genes. The 5.6 kb basis repeat unit which contains both - and -tubulin genes is repeated twenty times per haploid genome.Abbreviations SDS sodium dodecyl sulphate - 1×SSPE 150 mM NaCl, 10 mM NaH₂PO₄ and 1 mM EDTA, pH 7.4     

Larval development and food habits of the marbled parrotfish, <Emphasis Type="Italic">Leptoscarus vaigiensis</Emphasis>, associated with drifting algae

The larval development and food habits of the marbled parrotfish, Leptoscarus vaigiensis (Scaridae) associated with drifting algae were studied. In this study, 628 L. vaigiensis of various developmental stages ranging from postflexion larvae (9.4mm in standard length, SL) to adults (192.0mmSL) were sampled from drifting algae at two fishing ports in Nakagusuku Bay of Okinawa Island. In 3969 fish comprising 65 taxa in 34 families of Teleostei collected with drifting algae, L. vaigiensis occupied 15.8% of samples and occurred generally throughout the whole year. A large number of L. vaigiensis were collected from July to October accompanied by an occurrence of drifting algae composed of Sargassum spp. Larvae and early juveniles ranging from 11.1 to 14.9mmSL appeared sporadically throughout the year, and postflexion larvae 11mmSL occurred from July to November. Their food shifted from planktonic copepods in postflexion larvae and juveniles ranging from 10.0 to 14.9mmSL to seaweed in the juveniles ranging from 15.0 to 24.9mmSL. Furthermore, adults and young over 25mmSL fed almost exclusively on seaweed, with Sargassum spp. constituting the drifting algae. These facts indicate that drifting algae may have a role concerning food and habitat, and may act as a nursery for L. vaigiensis.     

Transposition of mobile genetic elements in interspecific hybrids of Drosophila

In situ hybridization of labeled DNA of four mobile dispersed genetic elements (mdg), isolated from D. melanogaster and C. virilis genomes, with polytene chromosomes of the larvae of several Drosophila species has been carried out. The data show that the mdg elements exhibit a high degree of species specificity. The same conclusions are derived from filter hybridization using ³²P-labeled D. melanogaster and D. virilis DNA and cloned mdg sequences immobilized on nitrocellulose filters. We attempted to induce transpositions (jumping) of mdg elements specific for D. virilis chromosomes to the chromosomes of related species (e.g. D. littoralis Meigen) originally lacking the representatives of this family of repeats. For this purpose we produced hybrid stocks with synthetic karyotoypes characterized by different combinations of D. virilis homologous chromosomes and hybrid chromosomes. In one of such stocks we did find by in situ hybridization the insertion of a D. virilis mdg element into the fifth chromosome of D. littoralis Meigen. The transposition (jumping) took place in the only region where somatic pairing between the fifth chromosomes of D. virilis and D. littoralis occurs more or less regularly in the hybrids. Since crossing-over in hybrid chromosomes of males is excluded in such synthetic stocks, gene conversion may be responsible for this transposition. The possible bearing of the phenomenon observed on the problem of hybrid dysgenesis is discussed.